1. Field of the Invention
This invention relates to an integrated heater and cooler on a microfluidic device for use in thermocycling, and more particularly, to a portable microfluidic card with a heating, cooling and heat cycling system on-board. This invention further relates to a microfluidic card having an integrated heat exchanger circuit, or thermal electric cooler (TEC) for use in connection with a microfluidic device to provide thermocycling for use in, for example, PCR or rtPCR.
2. Description of the Related Art
Integrated microfluidic handling systems that provide control over nanoliter sized volumes of liquid are useful in both miniaturizing present analytical tests and handling the small sample sizes frequently used in biomedical testing. Entire chemical analyses can be preformed on a single microfluidic device. The microfluidic devices include components such as channels, valves, pumps, flow sensors, mixing chambers and optical detectors. Examples of these components and systems may be found in U.S. Pat. Nos. 5,932,100; 5,922,210; 6,387,290; 5,747,349; 5,748,827; 5,726,751; 5,724,404; 5,716,852; 5,974,867; 6,007,775; 5,972,710; 5,971,158; 5,948,684; and 6,171,865 (which patents are hereby incorporated by reference in their entirety).
The ability to perform analyses microfluidically provide substantial advantages of throughput, reagent consumption, and automatability. Another advantage of microfluidic systems is the ability to integrate large numbers of different operations in a single “lab-on-a-chip” device for performing processing of reactants for analysis and/or synthesis. One example of an operation that would benefit from the advantages of microfluidics is the Polymerase Chain Reaction, commonly known as PCR or rtPCR, commonly known as reverse transcriptase-Polymerase Chain Reaction.
PCR is a technique used to amplify specific segments of DNA. In brief, DNA contacted with a solution containing theDNA polymerase, unbound nucleotide bases, and “primers” (i.e., short sequences of nucleotides that bind with an end of the desired DNA segment). Two primers are used. The first primer binds at one end of the desired segment on one of the two paired DNA strands, while the second primer binds at the other end but on the other DNA strand. The solution is heated to a temperature of about 95° C. to break the bonds between the strands of the DNA. Since the primers cannot bind the DNA strand at such high temperatures, the solution is cooled to about 55° C. At this temperature the primers bind or “anneal” to the separated strands. Since the DNA polymerase works best at around 72° C., the temperature is again raised and the DNA polymerase quickly builds a new strand by joining the free nucleotide bases to the primers. When this process is repeated, a strand that was formed with one primer binds to the other primer, resulting in a new strand that is restricted solely to the desired segment. Thus the region of DNA between the primers is selectively replicated. Further repetitions of the process can produce billions of copies of a small piece of DNA in several hours.
Enabling the detection of a specific bacterium or virus, or a genetic disorder, PCR has become one of the most powerful tools available for human diagnostics. Since PCR can amplify as little as a single molecule of DNA, problems of contamination become paramount. To minimize the risk of contamination, many laboratories have needed to set up separate rooms to house their PCR machines.
rtPCR is short for reverse transcriptase-polymerase chain reaction. It is a technique in which an RNA strand is transcribed into a DNA complement to be able to subject it to PCR amplification. Transcribing an RNA strand into a DNA complement is termed reverse transcription and is done by the enzyme reverse transcriptase.
PCR based assays have three basic steps: isolation of DNA, amplification of DNA, and detection of DNA. The DNA isolation process in the past involved very tedious procedures and was a limiting factor for diagnostic PCR. With advancement in technology, DNA isolation procedures have become simplified such that DNA can be quickly extracted with reagent addition and centrifugation. Although simplified, traditional methods of isolation require the use of expensive and cumbersome equipment, including for example a non-refrigerated centrifuge of at least 1300 rpm with relative centrifugal force (RCF) of about 16000 g is required since. In addition, a good autoclavable set of Micro-pipettes is also required for required for DNA extraction, as well as a variable speed heavy duty Vortex Mixer, a microwave oven for lysis of the cells, and a water bath for boiling and incubations.
After the DNA is isolated, a single DNA molecule can be amplified to as discussed above to more than a billion copies with the aid of a thermal cycler to change the temperatures from 96° C. to 55° C. to 72° C. in every cycle. In traditional PCR, use of glass capillaries as a reaction vessel for rapid heating and cooling of PCR reaction mixtures has been used to shorten the amplification time. However, even with these advancements, a system and method of PCR is needed that is simplified, minimizes the risk of contamination or human error, is portable, cost effective and accelerated. Once amplified, the DNA may be detected by any number of available techniques including, for example, with optical instruments. Detection of DNA can also be accomplished by electrophoresis or by liquid hybridization depending on whether confirmation or quantification is desired.
Although microfluidics has been used in a variety of applications, many technical issues with respect to performing the steps of isolation, amplification and detection remain for PCR to be effectively performed microfluidically. One difficulty is integration of a thermal cycler. Various attempts have been made to develop an adequate device for monitoring and changing the temperature on a microfluidic device. For example, International Patent Application PCT/US98/1791 is directed to a devices that controls and monitors temperature within microfluidic systems by applying electric currents to fluids to generate heat therein, as well as measure solution conductivity as a measure of fluid temperature.
Another system for controlling temperature on a microfluidic device is described in U.S. Pat. No. 6,541,274. This patent is directed to a reactor system having a plurality of reservoirs in a substrate. A heat exchanger is inserted in the reservoirs to control the temperature. Still others examples of existing devices for controlling temperature on a microfluidic device is with radiant heat as described in U.S. Pat. No. 6,018,616, and the temperature regulated controlled block as described in U.S. Pat. No. 6,020,187.
While significant advances have been made in the field of microfluidics generally, and PCR or rtPCR specifically, there remains a need in the art for microfluidic device that contains a thermal cycler, particularly in the context of microfluidic PCR or rtPCR. The present invention fulfils this need and provides further related advantages.